Botanical Studies (2012) 53: 243-254. MORPHOLOGY Stipules and colleters of the mangrove Rhizophoraceae: morphology, structure and comparative significance Chiou-Rong SHEUE1,*, Ying-Ju CHEN1, and Yuen-Po YANG2,3 1Department of Life Sciences, National Chung Hsing University, 250, Kuo Kuang Rd., Taichung 402, Taiwan 2Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 804, Taiwan 3Department of Bioresources, Dayeh University, 168 University Rd., Dacun, Changhua 515, Taiwan (Received October 11, 2010; Accepted December 1, 2011) ABSTRACT. One characteristic possessed by the entire mangrove Rhizophoraceae is the strong enclosure of its young shoots by conspicuous rounded or flattened stipules. Young leaves are conspicuously immersed in resinous to milky exudates from colleters (multicellular external secretory emergences) located at the adaxial stipule bases. We systematically studied and compared morphological and structural features of stipules and their colleters from 18 taxa of the mangrove Rhizophoraceae. Three types of sclereid idioblasts (Ceriops and Rhizophora), collenchymas, and thick cuticles, were found to provide a structural basis for the mechanical support of stipules. Several to hundreds (35-580) of finger-like colleters aggregate into genus-specific shapes: rectangular to trapezoidal (Bruguiera), triangular (Ceriops and Kandelia), or as a band (Rhizophora). Number of rows, total number of colleters per stipule, and their individual size, also vary by taxon, and have taxo- nomic value. All colleters of this subfamily are considered anatomically ‘standard’, regardless of whether they appear as short-stalked rods (Bruguiera), long-stalked rods (Ceriops, Kandelia) or acuminate rods (Rhizophora, no stalk). Colleters in these taxa are comprised of a central axis of slender, elongated cells and an outer pali- sade-like epidermis, with a secretory function, perpendicular to the axis. Based on stipule and colleter charac- teristics, Ceriops and Kandelia are closely related, but Rhizophora shows more derived features. The structural and mechanical protection provided by stipules and colleter exudates may help shield the young shoots of these mangrove plants from their harsh environments. Keywords: Anatomy; Bruguiera; Ceriops; Colleter; Kandelia; Mangroves; Rhizophora; Sclereid; Standard type; Stipule. INTRODUCTION the naked eye at the adaxial base of stipules in the species of mangrove Rhizophoraceae (Tomlinson, 1986; Sheue Rhizophoraceae is well known as the richest mangrove et al., 2003a, b; Sheue et al., 2005). Except for the genus family, having four exclusively mangrove genera (Bru- Bruguiera, colleters, although few in number, are also guiera, Ceriops, Kandelia and Rhizophora) (Tomlinson, present within the bracteoles of mangrove Rhizophoraceae 1986). About 21 mangrove species of this family are inflorescences (Sheue, 2003; Sheue et al., 2003a). currently recognized, including new taxa from Kandelia Colleters are external secretory structures (Fahn, 1990), (Sheue et al., 2003b) and Ceriops (Sheue et al., 2009a, b; characterized by the production of a viscous substance Sheue et al., 2010). The family is diversified both ecologi- that covers, protects from desiccation, and lubricates the cally (habitat) and morphologically (trait). In addition to bud and young leaves of plants (Kronestedt-Robards and the mangroves mentioned above, the family has 15 inland Robards, 1991; Evert, 2007). Hou (1958) mentioned that genera with 135 species occurring in inland forests (Jun- colleters in Rhizophoraceae were first observed by Al- cosa and Tomlinson, 1988a). All members of the Rhizo- ston in African Cassipourea, and later by Metcalfe and phoraceae s. s. (excluding Anisophyllea) have large and Chalk in Carallia (both inland genera). These glandular conspicuous interpetiolar, glabrous and caducous stipules emergences inside the adaxial regions of stipules appear on both vegetative and reproductive shoots, strongly to occur in all Malaysian genera, both mangrove and in- ensheathing the young leaves and inflorescences (Hou, land (Hou, 1858). Colleter exudates from this family are 1958). Several to hundreds of aggregated finger-like col- assumed to be composed of phenolic compounds (Rosh- leters mixed with milky mucilage can be observed with china and Roshchina, 1993). Colleters have long been associated with a number of * Corresponding author: E-mail: [email protected]; Tel/ flowering plants, notably Apocynaceae and Rubiaceae. The Fax: +886-4-22857395. color and consistency of colleter exudates are variable in 244 Botanical Studies, Vol. 53, 2012 a somewhat diagnostic manner from species to species of MATERIALS AND METHODS the mangrove Rhizophoraceae (Tomlinson, 1986). How- ever, the knowledge of colleters of Rhizophoraceae is still Plant materials containing stipules with colleters were under-reported and remains obscure. The presence of col- collected from 18 mangrove Rhizophoraceae taxa (includ- leters in R. mangle was noticed by Warming in 1883 and ing two hybrids of Rhizophora) from Asia and Australia briefly described (Gill and Tomlinson, 1969): “a feature of from 1999 to 2009 (Table 1). The voucher specimens were some physiological significance in the bud of Rhizophora deposited in the Herbarium of National Chung Hsing Uni- is a double row of yellow, glandular scales on the side of versity (TCB), Taichung, Taiwan. each stipule. The primodia enclosed by the stipules are Three individual plants were sampled from a popula- always bathed by a viscous fluid which seems to originate tion, and from each individual, three stipules were care- from these glands.” It is the only species in this family to fully detached from the shoot apices when the young have the colleters anatomically studied (Lersten and Cur- leaves were about to emerge from the stipules. Stipules tis, 1974). The main objectives of the present work are to were stored in 70% ethanol in the field, observed and pho- provide a systematic study of stipules and colleters of the tographed with a Zeiss dissecting microscope to determine mangrove species of this family in order to understand stipule morphology, number of colleters, and their forms their morphology, structure, taxonomic value, and com- of aggregation within the stipule. A portion of the materi- parative significance. als was observed with a S-2400 Scanning Electron Micro- Table 1. Taxa and collection information of the mangrove species of Rhizophoraceae in this study. Species Collection locality Habitat Bruguiera cylindrica (L.) Blume Pichavarum, Tamil Nadu, India Back or middle mangroves Sungei Buloh Wetland Reservoir, Singapore Bruguiera exaristata Ding Hou Cairns, Queensland, Australia Back or middle mangroves Bruguiera hainesii C. G. Rogers Pulau Ubin, Singapore Within mangroves Bruguiera gymnorhiza (L.) Savigny Sungei Buloh Wetland Reservoir, Singapore Middle mangroves Cardwell, Queensland, Australia Bruguiera parviflora (Roxb.) W. & A. ex Pasir Ris Nature Park, Singapore Back or middle mangroves Griff. Cardwell, Queensland, Australia Bruguiera sexangula (Lour.) Poir. Benut, Johor, Malaysia Middle mangroves Ceriops australis (C. T. White) Ballment, T. Cardwell, Queensland, Australia Back mangroves, landward J. Sm. & J. A. Stoddart Moreton Bay, Queensland, Australia Ceriops decandra (Griff.) Ding Hou Pichavarum, Tamil Nadu, India Back mangroves Ceriops pseudodecandra Sheue, H. Y. Liu, Darwin, Northern Territory, Australia Middle mangroves, along river bank C. C. Tsai and Yuen P. Yang Cairns, Queesland, Australia Back mangroves Ceriops tagal (Perr.) C. B. Rob. Sungei Buloh Wetland Reservoir, Singapore Within mangroves Cairns/ Cardwell, Queensland, Australia Back mangroves Ceriops zippeliana Blume Pasir Ris Nature Park, Singapore Back mangroves Kandelia candel (L.) Druce Benut, Johor, Malaysia Malaysia Along river bank Kandelia obovata Sheue, H. Y. Liu & J. W. Tanshui, Taipei, Taiwan Along river bank, pure stand H. Yong Tongshi, Chaiyi, Taiwan River bank Rhizophora apiculata Blume Sungei Buloh Wetland Reservoir, Singapore Seaward mangroves Pichavarum, Tamil Nadu, India Cairns/ Cardwell, Queensland, Australia Rhizophora × annamalayana Kathiresan Pichavarum, Tamil Nadu, India Back mangroves Rhizophora × lamarckii Montr. Cairns/ cardwell, Queensland, Australia Back mangroves Rhizophora mucronata Lam. Pichavarum, Tamil Nadu, India Seaward mangroves Sungei Buloh Wetland Reservoir, Singapore Rhizophora stylosa Griff. Tainan, Taiwan Along salty canal Cairns/ Moreton Bay, Queensland, Australia Seaward mangroves SHEUE et al. — Stipules and colleters of the mangrove Rhizophoraceae 245 scope (Hitachi, Tokyo, Japan), after treating with ethanol series dehydration, critical point drying and gold coat- ing, allowing shape and size determination in particular. These stipules were also treated with the clearing method (Chiang, 1990) to investigate features such as sclereid id- iobalsts and crystals. For field fixation for anatomical study, the base of each stipule with colleters was cut into several small pieces and put into 1.25-1.5% glutaraldehyde in 0.1 M phosphate buf- fer with 5% sucrose. Materials were stored in this fixative for 5-7 days during collection abroad. For the second fixa- tion, materials were subsequently transferred to 1% OsO4 in 0.1 M phosphate buffer for about 4 hours. After dehy- dration through an ethanol series, materials were infiltrated Figure 1. The mangrove Rhizophoraceae with shoots and for 3 days and embedded within Spurr’s-resin. The embed- stipules. (A) The shoot